The present invention relates to certain oligonucleotides, pharmaceutical agents containing these oligonucleotides, and the use thereof. The oligonucleotides are in particular able to inhibit the proliferation of pancreatic tumors. These oligonucleotides therefore have a therapeutic potential for the treatment of pancreatic tumors. This can involve, in the broadest sense, an antisense therapy.
Approximately 25,000 US citizens die from pancreatic cancer every year (A. Warshaw and C. Fernandez-del Castillo, New England J. Med. 326, 455 (1992)). Little is known about risk factors and the failure of radiation therapy and chemotherapy leads to a low 5-year survival rate after diagnosis.
Modified oligodeoxyribonucleotides (ODNs) have been found to be a group of very promising medicinal products in recent years and various mechanisms have been proposed for their mode of action. High expectations exist for the so-called antisense oligodeoxyribonucleotides (ASODNs) that suppress the expression of a specific protein, for example an oncogene.
Despite these high expectations and the in vitro demonstration that a specific gene is expressed to a lesser degree, only a single ASODN—Vitravene (ISIS, Carlsbad)—has been approved for use as a medicinal product.
On the other hand, oligodeoxyribonucleotides that do not have the sequence of an ASODN have been reported to have non-specific biological effects. The mechanism of action is not initially important for the development of a medicinal product, as long as the action can be clearly demonstrated in vivo and no side effects, or only justifiable ones, are seen.
Although a large number of modifications have been proposed and implemented in vitro for cell cultures (J. P. Shaw, K. Kent, J. Bird, J. Fischback, B. Froehler, Nucl. Acid Res. 19, 747 (1991), clinical studies and in vivo studies have been conducted almost exclusively with phosphorothioates in which an oxygen molecule has been replaced by a sulfur molecule in the phosphodiester linkages, on the assumption that such thioates will have a greater stability in the presence of nucleases, whilst being readily transported into cells. The half-life of normal phosphorodiester ODNs is on average 20-40 minutes, whereas the half-life of thioates is approximately 2-5 hours. This low increase in the stability of thioates may be the reason for the otherwise so promising model of antisense strategies in vivo not yet having led to a medicinal product. With a half-life of 2-5 hours, the bioavailability is too low and it remains to be seen what effects the shorter fragments, resultant upon degradation, will have.
It is known that ODNs with terminal modifications at the 3′ and 5′ position have a greater resistance to attack by exonucleases (M. Maier, K. Bleicher, H. Kalthoff, E. Bayer, Biomed. Peptd., Proteins & Nucl. Acids 1, 235 (1995)). Such modified ODNs have been used to date in cell cultures, but not in pre-clinical investigation models or clinical studies. In addition, most in vitro studies did not use phosphorothioates modified in the 3′ and 5′ positions, but instead the diester, so that endonucleases can still attack them. Lipophilic, cationic tensides, such as LipofektinR are often added in vitro to improve uptake by cells. Such methods have not been adopted in vivo, or are markedly limited in scope, for various reasons, including the toxicity of such adjuvants.
It has now been found that the proliferation of human pancreatic tumors can be suppressed in vitro in cell cultures and in vivo in orthotopic xenotransplantation models using certain oligodeoxyribonucleotides, and in particular those oligonucleotides that have covalently-bound groups in the terminal 3′ and/or 5′ positions. In particular it was found that those sequences rich in C and G, or at least one CG motive, are especially effective in inhibiting the proliferation of pancreatic tumors in vivo.